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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr10:89772129+89772239 111bp TTCTTTGCGTGGTGACTGAC AGAGAGAAGAGACAATGCTATGC
Mut= 119 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
ACAAGTGGTGCTTCTAGAGTTAAGTCAGAGTTTTAAAAAGTTACCCAGAGAATATCAAAATCAACTTCTAAATGGGATTGTGTAATTTCCTTCCTCAGAAAAGTTAAAAACACTATACGTCAGAATTTATTTCTGAAGTGTTTTGGGTTGGTTTCTAAGGCTGGGAGTAGCAGGTGATCCTCAGAGGAGTGGTATGACCTCTGACCCTTGTTCTTTGCGTGGTGACTGACATCTGTGCCATGTGAGCTGCTGTTAGagggtcctctcaatttccctacctttgtgtgcttggttaatagcatagcattgtctcttctctctgatagggtctacagtgaacaaaatgtggtagggactgggcttgcattgtgcctggaaagcagtggtggatacacagttttcatccatttgagggagtcaggaccagacttcagctgtttacaggaatgatgtacacatcggggcccccaaactgtagactcactattagagagttgtggcctggagagcagagaacagcagtgacagacactggacatgggttaaaataaactcatctctgtggtggagtttagtctgctgatgtgtttctagaactctgtatttgctaggttgtctgtagggtgcattagctgacactaaagtaatgtaagtgtggtctctttcagattcactaaaaacttatctcctgacaaaataaacctcagcacccttaaaggagagggggagctgaagaacctggagctggacgaggaggtgctgcagaacatgctggacctgcccacctggctggccatcagcaaggtgttctgcaataaagcctccatcagggtaagcaaggcctggctgtctgcggaggctgcctgctctcacaatagatactcggcttcaatagatACTCGGCTTCAGAACTGCTTATAAAGAAACACTGTGTTCCTAGTATTTACACAGAAAATAGCTGCATGAATGGTTTTCATCCCTTATGAGTATAACTGAAGTAACATATTAACAACTTACATTTACATTTTGTTTAAAACATCTTTATTGATAGTTTAATAAAATTGATTTATTATAAATTAATTGCTTTACTTTGGCATATATTCAATGATAGCCCGCCCCAGGTTTTATTGTCAAATGAAATGTTTTAAATTTTTAATACTT
This mutation is a 641 bp deletion beginning at Chromosome 10 position 89,772,175 bp and ending after 89,772,815 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48394 | TTC TTT GCG TGG TGA CTG AC | Common | A | |||
| 48395 | AGA GAG AAG AGA CAA TGC TAT GC | Wild type Reverse | A | |||
| 48396 | CCA TTC ATG CAG CTA TTT TCT GTG | Mutant Reverse | A | |||
| 48398 | Fluorophore-1 | TAG ACT CGG CTT CAG AAC TGC T | Quencher-1 | MUT Probe | ||
| 48405 | Fluorophore-2 | TCC TCT CAA TTT CCC TAC CTT TG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48394 | 0.40 uM |
| 48395 | 0.40 uM |
| 48396 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.